1. Field of the Invention
The present invention relates to a dry analytical element containing self-developing substrate for use in analysis of liquid such as body fluid.
2. Description of Prior Art
Self-developing substrates capable of dissociating p-nitrophenol pigment have been widely utilized in recent years. Particularly, the amylase analysis method using glucosidase enzyme and G5PNP or G7PNP obtained by introducing p-nitrophenol pigment (herein referred to as PNP) into oligosaccharide (such as G5 or G7) is superior in assay and accuracy to the conventional pigment starch method (for instance, Methods of Enzymatic Analysis, page 894, by H. U. Bergmeyer) in which a pigment starch substrate obtained by dyeing starch with a reactive dye, such as blue starch or amylopectin azure is used and assay is carried out by forming a precipitate by utilizing a difference in molecular weight distribution. Further, the amylase analysis method has an advantage in that the continuous monitoring assay (rate assay in a narrow sense) can be conducted.
However, p-nitrophenol pigment is a pH indicator and hence, its absorbance is greatly affected by the pH in reaction systems. Accordingly, there is a disadvantage that dissociation state greatly varies depending on pH.
For instance, pK (logarithm of the reciprocal of dissociation constant) of p-nitrophenol is 7.2 and less than 50% of p-nitrophenol released from the substrate is dissociated at a pH of about 7.0 which is the optimum pH for amylase (see, Clinical Pathology, extra number of June, No. 55, page 205 by Shin Takaya, written in the Japanese language).
Accordingly, in the measurement of amylase activity by using G5PNP or G7PNP, there is widely adopted a method wherein the released p-nitrophenol pigment is perfectly dissociated by a two-step reaction comprising (a) an enzymatic reaction and (b) photometric measurement after the termination of the enzymatic reaction by the addition of a solution having a high pH and photometry is conducted (by increasing detection sensitivity) at the sacrifice of the original continuous monitoring assay function.
Even when the amylase analysis method using glucosidase enzyme and the modified oligosaccharide obtained by introducing p-nitropenol into oligosaccharide, is used in the dry analytical element, there are serious disadvantages that the dissociation of p-nitrophenol released by the amylase reaction and the glucosidase reaction is not satisfactory and sensitivity is low. Particularly, in integral multilayer analytical elements, layers are in fluid contact with each other and the pH of these layers are practically equal so that it has been difficult to meet the requirements for the optimum pH for amylase and glucosidase as well as the satisfactory dissociation of p-nitrophenol. Further, the method using the aforementioned two-step reaction severely damages the simplicity and speediness which are advantages claimed for the dry analytical method.
In the measurement of the activity of acid phosphatase by using p-nitrophenyl phosphate as a self-developing substrate, released p-nitrophenol is not dissociated at all and color formation does not occur, when the reagent layer of the integral multilayer analytical element buffers at a pH of 4 which is the optimum pH for enzyme. When the reagent layer is kept at a pH range within which p-nitrophenol is dissociated and develops a color, that is, the reagent layer is kept at a pH of 7.2 or above, the dissociation of p-nitrophenol from the substrate by enzyme does not proceed and p-nitrophenol does not develop a color in a short time.